Despite extensive research on malaria for over a century, critical gaps remain in our understanding of the vectors; gaps that also limit our success in malaria control. We address some of the most critical gaps, namely the strategies used by African malaria mosquitoes to persist through the long dry season without surface waters for several months. Combining field and laboratory studies, our results provide compelling evidence that malaria vectors persist locally through the dry season by a form of dormancy (aestivation) and also engage in wind-assisted long-distance migration probably over hundreds of kilometers. These fundamental facets of vector biology have been controversial and, until now, were ignored due to insufficient evidence. Conventional and novel malaria and vector control strategies cannot afford to ignore aestivation and long-distance migration as processes that may hinder or aid the ultimate control of the disease. This year, we have published a study on the seasonal and spatial variation in microbiome composition in Sahelian and riparian A. coluzzii. The 16S bacterial data consisted 426 genera that revealed several compositional differences that were seasonally and spatially linked. These seasonal shifts were primarily in Ralstonia, Sphingorhabdus, Duganella and - Anaplasma, which was uniquely found In Sahelian dry season mosquitoes. This study supports a physiological or ecological switch in bacterial composition in relation to aestivation and identifies a few putative taxa as markers. However, additional studies are needed to confirm these results; the genomic analysis underway may provide such opportunity (below). To address limitations of our recent genetic study to trace the origin of A. coluzzii wet-season populations in the Sahel (Lehmann et al. 2017), we have collaborated with the Sanger Center (UK), where whole genome sequencing on >1000 mosquitoes, representing 12 time points over four years we provided is carried out. This genomic dataset, once available, will be used to comprehensively address the origin of the dry season population and other critical questions. This genomic data may also be useful to test resiliency of the findings in microbiome study (above). This year, we have carried out two novel studies using marking natural mosquitoes of mosquitoes with stable isotopes. Following a laboratory experiments in LMVR, our first field study in Mali aimed to test and optimize the methodology for marking adult mosquitoes with deuterium by enrichment of water and microbiota in natural larval sites. Our results demonstrated sound capacity to mark adult mosquitoes under field conditions using the optimized protocol and that the enrichment in these mosquitoes were detected up to their death (up to 6 weeks). Building on these results, a second field experiment started in two villages in the Sahel, aiming to quantify the importance of aestivation in the persistence of malaria mosquitoes. Preliminary results reveal a dramatic picture. By early November 2017, when the last larval sites dried up, we marked 50% of the adult mosquitoes in these villages. Sampling of adults continued throughout the dry season (December-May) and immediately after the first rains June-July 2018. IRMS analysis has revealed that 25-40% of the mosquitoes appearing in March during the late dry season peak had levels of 2H above natural levels, i.e., they grew up as larvae in the enriched larval sites 4 months earlier. These exciting results, prove that (i) A. coluzzii appearing during the late dry season peak consists of aestivators, rather than long-distance migrators, (ii) since the first rain typically falls 1-2 months later, the survival of local mosquitoes for 4 months strongly supports aestivation as the main persistence strategy of A. coluzzii, and (iii) this novel way of marking mosquitoes is powerful to elucidate other critical problems in vector-borne disease. Additional mosquitoes will be analyzed by IRMS in the coming months including more mosquitoes from the dry season and those collected after the first rains (soon to be shipped from Mali to LMVR). The first paper describing the methodology we developed for marking wild African malaria mosquitoes is being written. Experiments to simulate aestivation in the laboratory has have yielded encouraging results, with a combination of photoperiod, temperature, and priming of larvae leading to survival of A. gambiae s.l. over 100 days. These results are now being replicated with subsamples assays for variation in transcription, stress tolerance, and lipid content to further test the relation of this long-lived phenotype with aestivation. A transcriptomic analysis of mosquitoes samples representing different seasons from the Sahel and perennial area in the wet savanna of Mali is underway. Data analysis and writing is in progress on aerial sampling of mosquitoes flying in high altitudes (40-290 m above ground) using traps tethered to helium filled balloons. Using NOAA's HYSPLIT meteorological models, we track mosquitoes backwards to infer flight trajectories and putative source populations. With this tool, we can complete the analysis and writing of at least 3 publications. In additional to anopheline mosquitoes, we collected approximately 2,500 culicine mosquitoes, many of which might be vectors of arboviruses. Using COI barcode analysis carried out in our lab and in the Walter Reed Biosystematics Unit by Yvonne Lintons group. The identification has recently been completed. Members of >40 mosquito species with notable pathogen vectors including plasmodia, filariae, and arboviruses (e.g., Rift Valley Fever, West Nile). Among the insects collected in high altitude, we identified and quantified pest species in agriculture affecting food security, such as leafhoppers vectors of viruses and bacteria to rice, maize, sorghum, and millet. These are also analyzed to demonstrate the value of this aerial collection and linked data to the scientific community and encourage members to use the collection their own independent studies. Our first manuscript on diverse insect taxa collected in high altitudes has been completed and is circulating among authors and friendly reviewers. Data analysis and writing is being completed on a study on flight aptitude of >1200 wild mosquitoes bio-assayed in Mali. The results reveal that mosquitoes fly over the 9 hours night assay, with maximum total flight of 5 hours. Variation between species over different seasons and relationships to morphometric traits may help clarify if some of the flights represent long distance migration. In summary, we carry out a set of complementary novel studies to address the roles of dormancy and windborne migration in African mosquito vectors.